PROJECT SUMMARY The identification of TET2 mutations and the subsequent discovery of DNMT3a and IDH1/2 mutations in myeloid malignancies has led to the realization that dynamic changes in DNA methylation patterns induced by somatic mutations are critical to hematopoietic transformation. The TET proteins are iron/?-ketoglutarate (Fe++/?-KG)-dependent dioxygenases and are able to modify 5-methylcytosine (5mC) on DNA to 5- hydroxy/formyl/carboxly-cytosine (5hmC, 5fC, 5caC), which leads to subsequent DNA demethylation. TET2 is targeted by somatic mutations in a spectrum of myeloid malignancies, including 10-20% of AML. Our laboratories have led studies characterizing the mutational spectra of TET2 in myeloid leukemias and in in vivo studies demonstrating that TET2 is a haploinsufficient tumor suppressor, which increases hematopoietic stem cell self-renewal and myeloid transformation. Supported by this award we were able to study in detail TET2 function in leukemia. We identified TET2 interacting proteins, generated disease models by modeling Tet2 alterations in concert with co-occurring myeloid disease alleles, delineated the relative role of Tet1 and Tet2 in hematopoietic function, employed techniques to map TET2-mediated 5hmC deposition at a genome-wide scale, and restored Tet2 activity in animal models and identified compounds that can target TET2-mutant myeloid leukemia. Moreover, we were able to show that TET2 mutations are an initiating event in clonal hematopoiesis (CH) that then ?seeds? the ground for additional somatic alterations, which then drive progression to myeloid transformation. This places us in an excellent position to address significant new questions relating to the molecular roles of TET2 in the initiation and progression of myeloid leukemia: 1) Are mutational order and/or cell compartment which acquires TET2/cooperating mutations critical in myeloid transformation? 2) Can we suppress disease progression, even in the presence of additional mutational events, if we restore wild-type TET2 expression and function? 3) Are there functional differences between TET2 loss and TET2 missense mutations that are seen in a subset of leukemia patients? We will address these important questions through the use of state-of-the art mouse models, epigenomic profiling techniques, and studies in primary patient samples to elucidate novel mechanisms of TET2-mediated transformation.